Modified adenoviral fiber and target adenoviruses

ABSTRACT

The invention relates to an adenovirus fiber modified by the mutation of one or more residues. The residues are directed towards the natural cell receptor in the three-dimensional structure of said adenovirus. The invention further relates to a DNA fragment, and expression vector, and a cell line expressing said fiber, and also concerns an adenovirus, the process for producing this type of adenovirus, and a infectable host cell, as well as their therapeutic application and a corresponding pharmaceutical composition.

The subject of the present invention is an adenoviral fiber mutated inthe regions involved in the recognition and the binding of the naturalcell receptor for adenoviruses. It also relates to the recombinantviruses carrying at their surface such a fiber and a ligand whichconfers on them a modified or targeted host specificity towards aparticular cell type, the cells containing these adenoviruses as well asa method for preparing infectious viral particles thereof intended fortherapeutic use. The invention is most particularly of interest for genetherapy perspectives, in particular in humans.

By virtue of their particular properties, adenoviruses are used in anincreasing number of applications in gene therapy. Having beenidentified in numerous animal species, they are not very pathogenic, arenonintegrative and replicate both in dividing and quiescent cells.Furthermore, they exhibit a broad host spectrum and are capable ofinfecting a very large number of cell types such as epithelial cells,endothelial cells, myocytes, hepatocytes, nerve cells and synoviocytes(Bramson et al., 1995, Curr. Op. Biotech. 6, 590-595). However, thisabsence of specificity of infection could constitute a limit to the useof recombinant adenoviruses, on the one hand, from a safety point ofview since there may be dissemination of the recombinant gene in thehost organism and, on the other hand, from the efficiency point of viewsince the virus does not infect specifically the cell type which it isdesired to treat.

In general, the adenoviral genome consists of a double-stranded linearDNA molecule of about 36 kb containing the genes encoding the viralproteins and, at its ends two inverted repeats (designated ITR forInverted Terminal Repeat) involved in the replication and theencapsidation region. The early genes are distributed in 4 regionsdispersed in the adenoviral genome (E1 to E4; E for early), containing 6transcriptional units equipped with their own promoters. The late genes(L1 to L5; L for late) partly cover the early transcription units andare, for the most part, transcribed from the major late promoter MLP.

As a guide, all the adenoviruses used in gene therapy protocols aredeficient for replication by deletion of at least the E1 region and arepropagated in a complementation cell line which provides in trans thedeleted viral functions. The 293 line, established from human embryonickidney cells, which efficiently complements the E1 function (Graham etal., 1977, J.

Gen. Virol. 36, 59-72), is commonly used. Second-generation vectors haverecently been proposed in the literature. They conserve the regions incis which are necessary for the replication of the virus in the infectedcell (ITRs and encapsidation sequences) and contain substantial internaldeletions designed to eliminate most of the viral genes whose expressionin vivo can lead to the establishment of inflammatory or immuneresponses in the host. The adenoviral vectors and the technique fortheir preparation have been the subject of numerous publications whichare accessible to persons skilled in the art.

The infectious cycle for adenoviruses occurs in 2 steps. The early phaseprecedes the initiation of replication and makes it possible to producethe early proteins regulating the replication and transcription of theviral DNA. The replication of the genome is followed by the late phaseduring which the structural proteins which constitute the viralparticles are synthesized. The assembly of the new virions takes placein the nucleus. In a first stage, the viral proteins assemble so as toform empty capsids of icosahedral structure into which the genome isencapsidated. The adenoviruses liberated are capable of infecting otherpermissive cells. In this regard, the fiber and the penton base presentat the surface of the capsids play a critical role in the cellularattachment of the virions and their internalization.

The adenovirus binds to a cellular receptor present at the surface ofthe permissive cells via the trimeric fiber (Philipson et al., 1968, J.Virol. 2, 1064-1075; Defer et al., 1990, J. Virol. 64, 3661-3673). Theparticle is then internalized by endocytosis through the binding of thepenton base to the cellular integrins α_(v)β₃ and α_(v)β₅ (Mathias etal., 1994, J. Virol. 68, 6811-6814). The capacity of the soluble fiberor of anti-fiber antibodies to inhibit infection demonstrates its rolein the cellular attachment of the virus.

The fiber is composed of 3 domains (Chroboczek et al., 1995, CurrentTop. Microbiol. Immunol. 199, 165-200):

(1) At the N-terminus, the tail, which is highly conserved from oneserotype to another, interacts with the penton base and ensures theanchorage of the molecule in the capsid.

(2) The stem is a structure in the form of a rod, composed of a numberof repeats of β sheets, this number varying depending on the serotypes.

(3) Finally, at the distal end of the stem, the head is a sphericalglobular structure which contains the trimerization signals (Hong andEngler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J.Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 185,365-376). Furthermore, most of the experimental data show that the headdomain is responsible for the binding to permissive cells (Henry et al.,1994, J. Virol. 68, 5239-5246; Louis et al., 1994, J. Virol. 68,4104-4106).

“Targeted” adenoviruses whose native fiber is modified so as torecognize a different cellular receptor have already been proposed inthe literature. Thus, WO94/10323 describes mutants of the fiber of Ad5,into which a sequence encoding an antibody fragment (of the scFv type)is inserted at the end of one of the 22 repetitive units of the stemwith the aim of modifying the specificity of infection towards cellshaving the target antigen. U.S. Pat. No. 5,543,328 describes an Ad5chimeric fiber in which the head domain is replaced by tumor necrosisfactor (TNF) so as to interact with the cellular receptor for TNF. Inanother construct, the Ad5 native fiber is fused at its C-terminal endwith the peptide ApoE allowing binding to the LDL (for low densitylipoprotein) receptor present at the surface of hepatic cells.WO95/26412 describes a fiber modified by incorporation of a ligand atthe C-terminal end which conserves its trimerization capacities.WO96/26281 describes a chimeric fiber obtained by replacing part of thenative fiber and, in particular, the head, with the equivalent part ofan adenoviral fiber of another serotype and, optionally, by inserting atthe C-terminal end a peptide RGD which is specific for vitronectin.

As indicated above, the specificity of infection of an adenovirus isdetermined by the attachment of the adenoviral fiber to a cellularreceptor situated at the surface of permissive cells.

French patent application 97 01005 has identified the role of theantigens of the class I major histocompatibility complex and of the IIImodules of fibronectin as primary receptor and as cofactor,respectively, for adenoviruses. However, other proteins may be involved.In this regard, recent studies have presumed the use of the cellularreceptor for the coxsackie viruses by the types 2 and 5 adenoviruses topenetrate into their target cells (Bergelson et al., 1997, Science 275,1320-1323). The problem which the present invention proposes to solve isto modify the region for interaction of the adenoviral fiber with thecellular receptor(s) in order to alter the natural host specificity ofthe adenoviruses carrying the mutated fiber. For ease of understanding,the term “cellular receptor” for adenoviruses will be used hereinafterto designate the cellular polypeptide(s) involved directly or otherwisein the binding of adenoviruses to their natural target cells or in thepenetration into the latter. Of course, said receptor may be differentdepending on the serotypes. The addition of a ligand makes it possibleto confer a new tropism toward one or more specific cell types carryingat their surface a target molecule recognized by the ligand in question.

The present invention constitutes an improvement of the previoustechnique since it discloses the regions of the fiber to be mutated inorder to inhibit or prevent binding to the natural cellular receptor foradenoviruses. One or more residues of the 443 to 462 region of the headof the Ad5 fiber have now been substituted or deleted and an inhibitionof the infectivity of the corresponding adenoviruses toward normallypermissive cells has now been shown. The introduction of the GRP (forgastrin releasing peptide) ligand into these fibers should make itpossible to target the infection toward cells expressing the GRPreceptor. The aim of the present invention is to reduce the therapeuticquantities of adenoviruses to be used and to target the infection at thecells to be treated. This specificity is essential when an adenovirusexpressing a cytotoxic gene is used, in order to avoid the propagationof the cytotoxic effect to healthy cells. The advantages offered by thepresent invention are to reduce the risks of dissemination and thesecondary effects linked to the adenoviral technology.

Accordingly, the subject of the present invention is an adenovirus fibermodified by mutation of one or more residues of said fiber,characterized in that said residues are directed toward the naturalcellular receptor for said adenovirus.

The term “fiber” is widely defined in the introductory part. The fiberof the present invention may be derived from an adenovirus of human,canine, avian, bovine, murine, ovine, porcine or simian origin or may bea hybrid and may comprise fragments of diverse origins. As regards humanadenoviruses, the use of those of serotype C and, in particular, thetype 2 or 5 adenoviruses (Ad2 or Ad5) is preferred. It is indicated thatthe Ad2 fiber contains 580 amino acids (aa) whose sequence is disclosedin Herissé et al. (1981, Nucleic Acid Res. 9, 4023-4042). That of Ad5has 582 aa and its sequence, presented in the sequence identifier 1 (SEQID NO: 1) has been determined by Chroboczek and Jacrot (1987, Virology161, 549-554). When the fiber of the present invention originates froman animal adenovirus, bovine adenoviruses and, in particular, those ofthe BAV-3 strain are preferably used. The latter have been the subjectof numerous studies and the sequence of the fiber is disclosed ininternational application WO95/16048. Of course, the fiber of thepresent invention may exhibit other modifications compared to the nativesequence, other than those which are the subject of the presentinvention.

In accordance with the aims pursued by the present invention, the fiberaccording to the invention is modified so as to reduce or abolish itscapacity to bind to the natural cellular receptor. Such a property maybe verified by studying the infectivity or the cellular binding of thecorresponding viruses by applying the techniques of the art such asthose detailed below. According to an advantageous embodiment, thetrimerization and penton-base-binding properties are not affected.

For the purposes of the present invention, the term “mutation”designates a deletion, substitution or addition of one or more residuesor a combination of these possibilities. The case where the regions forinteraction with the natural cellular receptor are deleted completely orpartly and replaced in particular with a ligand specific for a cellsurface protein other than the natural receptor for adenoviruses is mostparticularly preferred.

The three-dimensional crystallographic structure of the adenoviral headhas been determined by Xia et al. (1994, Structure 2, 1259-1270). Eachmonomer contains 8 antiparallel β sheets designated A to D and G to Jand 6 major loops of 8 to 55 residues. For example, the CD loop linksthe β sheet C to the β sheet D. It is indicated that the minor sheets Eand F are considered to be part of the DG loop situated between the Dand G sheets. As a guide, Table 1 indicates the location of thesestructures in the amino acid sequence of the fiber of Ad5 as shown inthe sequence identifier No. 1 (SEQ ID NO: 1), +1 representing theinitiator Met residue. In general, the sheets form an ordered andcompact structure whereas the loops are more flexible. These terms areconventional in the field of protein biochemistry and are defined inbasic manuals (see for example Stryer, Biochemistry, 2nd edition, Chap2, p 11 to 39, Ed. Freeman and Company, San Francisco).

TABLE 1 β sheet loop nomenclature residues nomenclature residues A 400to 403 AB 404 to 418 B 419 to 428 — — C 431 to 440 CD 441 to 453 D 454to 461 DG 462 to 514 G 515 to 521 GH 522 to 528 H 529 to 536 HI 537 to549 I 550 to 557 IJ 558 to 572 J 573 to 578

The four β sheets A, B, C and J constitute the V sheets directed towardthe viral particle. The other four (D, G, H and I) form the R sheets,which are supposed to face the cellular receptor. The V sheets appear toplay an important role in the trimerization of the structure whereas theR sheets are thought to be involved in the interaction with thereceptor. The residues of the fiber of Ad2, Ad3, Ad5, Ad7, Ad40, Ad41and of the canine adenovirus CAV forming these different structures areclearly indicated in the preceding reference.

The modifications of the adenoviral fiber according to the inventionaffect more particularly the domain extending from the CD loop to the Isheet and involve in particular residues 441 to 557 of the Ad5 fiber and441 to 558 of the Ad2 fiber. As a result of their spatial location inthe native fiber, these residues are capable of recognizing and/orinteracting directly or indirectly with the natural cellular receptorfor the adenovirus in question. Inside this region, it is preferable tomodify the part which comprises the CD loop, the D sheet and theproximal part of the DG loop (positions 441 to 478 of the Ad2 and Ad5fiber) and, more particularly, the region extending from residues 443 to462 as regards Ad5 or 451 to 466 in the case of Ad2. The other targetregion for the modifications is the H sheet (aa 529 to 536 of the Ad5fiber and of the Ad2 fiber). Another alternative consists in themodification of the minor sheets E (aa 479-482 Ad5) and F (aa 485-486Ad5).

As indicated above, it is possible to carry out the procedure bysubstituting one or more amino acids in the regions exposed. There maybe mentioned in this regard the following examples which are derivedfrom the Ad5 fiber in which:

the glycine residue at position 443 is substituted by an aspartic acid,

the leucine residue at position 445 is substituted by a phenylalanine,

the glycine residue at position 450 is substituted by an asparagine,

the threonine residue at position 451 is substituted by a lysine,

the valine residue at position 452 is substituted by an asparagine,

the alanine residue at position 455 is substituted by a phenylalanine,

the leucine residue at 457 is substituted by an alanine or a lysine,and/or

the isoleucine residue at position 459 is substituted by an alanine.

It is also possible to introduce several substitutions into the targetedregion of the fiber, in particular at the level of the amino acidsforming an elbow, preferably of the αα type (see Table 2 by Xia et al.,1994, supra). By way of illustration, there may be mentioned thefollowing two examples in which the Ad5 fiber is modified bysubstitution:

of the glycine residue at position 443 by an aspartic acid,

of the serine residue at position 444 by a lysine, and

of the alanine residue at position 446 by a threonine; or

of the serine residue at position 449 by an aspartic acid,

of the glycine residue at position 450 by a lysine,

of the threonine residue at position 451 by a leucine, and

of the valine residue at position 452 by a threonine.

Of course, the replacement amino acids are only mentioned as guide andany amino acid may be suitable for the purposes of the presentinvention. It is preferable, nevertheless, not to drastically modify thethree-dimensional structure. Preferably, the amino acids forming anelbow will be replaced by residues forming a similar structure such asthose described in the Xia et al. reference already mentioned.

The fiber of the present invention may also be modified by deletion. Theregion eliminated may involve all or part of the domain exposed and, inparticular, of the CD loop, of the D sheet, of the DG loop and/or of theE and F sheets. As regards an Ad5 fiber according to the invention,there may be mentioned more particular the deletion:

of the region extending from the serine at position 454 to thephenylalanine at position 461,

of the region extending from the valine at position 441 to the glutamineat position 453,

of the region extending from the valine at position 441 to thephenylalanine at position 461, or

of the region extending from the asparagine at position 479 to thethreonine at position 486.

It is also possible to generate other mutants from substitution ordeletion in the other sheets or loops, such as for example the G, H andI sheets and the HI and DG loops.

According to an advantageous embodiment, when at least one of themodifications is a deletion of at least 3 consecutive residues of a loopand/or of a sheet, the deleted residues may be replaced by residues ofan equivalent loop and/or sheet derived from a fiber of a secondadenovirus capable of interacting with a cellular receptor differentfrom that recognized by the first adenovirus. The second adenovirus maybe of any origin, human or animal. This makes it possible to maintainthe structure of the fiber according to the invention while conferringon it a host specificity corresponding to that of the second adenovirus.As indicated in Xia et al. (1994, supra), the cellular receptormediating the infection by types 2 and 5 adenoviruses is different fromthat interacting with the types 3 and 7 adenoviruses. Thus, an Ad5 orAd2 fiber deleted for at least 3 consecutive residues among thosespecified above may be substituted by the residues derived from anequivalent region of the Ad3 or Ad7 fiber in order to reduce itscapacity to bind the Ad5 receptor and confer on it a new specificitytoward the cellular receptor for Ad3 or Ad7. By way of nonlimitingexample, there may be mentioned the replacement of theLAPISGTVQSAHLIITRFD (SEQ ID NO: 41) residues (positions 445 to 462) ofthe Ad5 fiber with the VNTLFKNKNVSINVELYFD (SEQ ID NO: 42) residues ofthe Ad3 fiber of the replacement of the PVTLTITL (SEQ ID NO: 43)residues (position 529 to 536) of the fiber of Ad5 with the PLEVTVML(SEQ ID NO: 44) residues of the fiber of Ad3.

The present invention also relates to an adenovirus fiber having asubstantially reduced capacity for binding to the natural cellularreceptor and nevertheless capable of trimerizing and of binding thepenton base. As indicated above, the natural cellular receptor isadvantageously chosen from the group consisting of the class I majorhistocompatibility antigens, fibronectin and the cellular receptor forthe coxsackie viruses (CAR) or any other cell surface determinant whichis usually involved or which participates in the infectivity ofadenoviruses.

According to an equally advantageous embodiment, the fiber according tothe invention comprises, in addition, a ligand. For the purposes of thepresent invention, the term ligand defines any entity capable ofrecognizing and binding, preferably with a high affinity, a cell surfacemolecule different from the natural cellular receptor. This molecule maybe expressed or exposed at the surface of the cell which it is desiredto target (cell surface marker, receptor, antigenic peptide presented byhistocompatibility antigens and the like). In accordance with the aimspursued by the present invention, a ligand may be an antibody, apeptide, a hormone, a polypeptide or a sugar. The term antibodycomprises in particular monoclonal antibodies, antibody fragments (Fab)and single-chain antibodies (scFv). These names and abbreviations areconventional in the field of immunology.

Within the framework of the present invention, it may be advantageous totarget more particularly a tumor cell, an infected cell, a particularcell type or a category of cells carrying a specific surface marker. Forexample, if the host cell to be targeted is a cell infected with the HIVvirus (Human Immunodeficiency Virus), the ligand may be a fragment ofantibody against fusin, the CD4 receptor or against an exposed viralprotein (envelope glycoprotein) or the part of the TAT protein of theHIV virus extending from residues 37 to 72; (Fawell et al., 1994, Proc.Natl. Acad. Sci. USA 91, 664-668). As regards a tumor cell, the choicewill be on a ligand recognizing an antigen specific for tumors (forexample the MUC-1 protein in the case of breast cancer, some epitopes ofthe E6 or E7 proteins of the papillomavirus HPV) or overexpressed(receptor for IL-2 overexpressed in some lymphoid tumors, GRP peptide,for Gastrin Releasing Peptide, overexpressed in lung carcinoma cells(Michael et al., 1995 Gene Therapy 2, 660-668) and in pancreas, prostateand stomach tumors). If it is desired to target the T lymphocytes, it ispossible to use a ligand for the T cell receptor. Moreover, transferrinis a good candidate for hepatic screening. In general, the ligands whichmay be used in the context of the invention are widely described in theliterature and may be cloned by standard techniques. It is also possibleto synthesize them by the chemical route and to couple them to the fiberaccording to the invention. In this regard, the coupling of galactosylresidues should confer a hepatic specificity because of the interactionwith the asialoglycoprotein receptors. However, the preferred embodimentconsists in inserting the ligand at the C-terminal end of the fiberaccording to the invention or as a replacement for the deleted residueswhen at least one of the modifications is a deletion of at least 3consecutive residues.

The present invention also relates to a DNA fragment encoding a fiberaccording to the invention as well as to a vector for expressing such afragment. Any type of vector may be used to this effect, whether it isof plasmid or viral origin, integrative or otherwise. Such vectors arecommercially available or are described in the literature. Likewise,persons skilled in the art are capable of adapting the regulatoryelements necessary for the expression of the DNA fragment according tothe invention. In addition, it may be combined with one or moresubstances capable of improving the transfection efficiency and/or thestability of the vector. These substances are widely documented in theliterature accessible to persons skilled in the art (see for exampleFelgner et al., 1989, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgsonand Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994,Bioconjugate chemistry 5, 647-654). By way of nonlimiting illustration,these may be polymers, lipids, in particular cationic lipids, liposomes,nuclear proteins and neutral lipids. A combination which can beenvisaged is a vector combined with cationic lipids (DC-Chol, DOGS andthe like) and neutral lipids (DOPE).

The present invention also relates to an adenovirus lacking a functionalnative fiber and which comprises, at its surface, a fiber according tothe invention. The latter may be expressed by the adenoviral genome orprovided in trans by a complementation cell line, such as those definedbelow. It may, in addition, comprise a ligand as defined above.Preferably, the specificity of binding of such an adenovirus to itsnatural cellular receptor is significantly reduced or, even better,abolished, because of the modified fiber which it carries. The loss ofthe natural specificity may be evaluated by studies of cellularattachment carried out in the presence of labeled viruses (for examplelabeled with ³H-thymidine according to the technique of Roelvink et al.,1996, J. Virol. 70, 7614-7621) or by studies of infectivity of cellswhich are permissive or which express the surface molecule targeted bythe ligand (see the examples which follow).

The ligand may be chemically coupled to the adenovirus according to theinvention. However, the variant according to which the sequencesencoding the ligand are inserted into the adenoviral genome, inparticular, into sequences encoding the modified fiber according to theinvention, preferably, in phase in order to preserve the reading frame,is preferred. The insertion may take place at any site. However, thepreferred site of insertion is upstream of the stop codon at theC-terminal end or in place of the deleted residues. It is also possibleto envisage introducing the sequences of the ligand into otheradenoviral sequences, in particular those encoding another capsidprotein, such as the hexon or the penton.

Advantageously, an adenovirus according to the invention is recombinantand replication-defective, that is to say incapable of autonomouslyreplicating in a host cell. The deficiency is obtained by mutation ordeletion of one or more essential viral genes and, in particular, of allor part of the E1 region. Deletions in the E3 region may be envisaged inorder to increase the cloning capacities. However, it may beadvantageous to conserve the sequences encoding the gp19 k protein(Gooding and Wood, 1990, Critical Reviews of Immunology 10, 53-71) inorder to modulate the immune responses of the host. Of course, thegenome of an adenovirus according to the invention may also compriseadditional deletions or mutations affecting other regions, in particularthe E2, E4 and/or L1-L5 regions (see for example internationalapplication WO94/28152 and Ensinger et al., 1972, J. Virol. 10, 328-339describing the heat-sensitive mutation of the DBP gene of E2).

According to a preferred embodiment, an adenovirus according to theinvention is recombinant and comprises one or more genes of interestplaced under the control of the elements necessary for their expressionin a host cell. The gene in question may be of any origin, genomic, cDNA(complementary DNA) or hybrid (minigene lacking one or more introns). Itmay be obtained by conventional molecular biology techniques or bychemical synthesis. It may encode an antisense RNA, a ribozyme or anmRNA which will then be translated into a polypeptide of interest. Thelatter may be cytoplasmic, membranal or may be secreted from the hostcell. Moreover, it may be all or part of a polypeptide as found innature, a chimeric polypeptide obtained from the fusion of sequences ofdiverse origins, or of a polypeptide mutated relative to the nativesequence having improved and/or modified biological properties.

In the context of the present invention, it may be advantageous to usethe genes encoding the following polypeptides:

cytokines or lymphokines (α-, β- and γ-interferons, interleukins and inparticular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factors (TNF),colony stimulating factors (GM-CSF, C-CSF, M-CSF and the like);

cellular or nuclear receptors, in particular those recognized bypathogenic organisms (viruses, bacteria or parasites) and, preferably,by the HIV virus or their ligands (fas ligand);

proteins involved in a genetic disease (factor VII, factor VIII, factorIX, dystrophin or minidystrophin, insulin, CFTR protein (Cystic FibrosisTransmembrane Conductance Regulator), growth hormones (hGH);

enzymes (urease, renin, thrombin and the like);

enzyme inhibitors (α1-antitrypsin, antithrombin III, viral proteaseinhibitors and the like);

polypeptides with antitumor effect which are capable of at leastpartially inhibiting the initiation or the progression of tumors orcancers (antibodies, inhibitors acting on cell division or transductionsignals, products of expression of tumor suppressor genes, for examplep53 or Rb, proteins stimulating the immune system and the like);

proteins of the class I or II major histocompatibility complex orregulatory proteins acting on the expression of the corresponding genes;

polypeptides capable of inhibiting a viral, bacterial or parasiticinfection or its development (antigenic polypeptides having immunogenicproperties, antigenic epitopes, antibodies, transdominant variantscapable of inhibiting the action of a native protein by competition andthe like);

toxins (herpes simplex virus 1 thymidine kinase (HSV-1-TK), ricin,cholera toxin, diphtheria toxin and the like) or immunotoxins,

markers (β-galactosidase, luciferase and the like),

polypeptide having an effect on apoptosis (inducer of apoptosis: Bax andthe like, inducer of apoptosis Bcl2, Bclx), cytostatic agents (p21, p16,Rb and the like), apolipoproteins (apoE and the like), SOD, catalase,nitric oxide synthase (NOS); and

growth factors (FGF for Fibroblast growth Factor, VEGF for VascularEndothelial, cell growth Factor and the like).

It should be noted that this list is not limiting and that other genesmay also be used.

Moreover, an adenovirus according to the invention may, in addition,comprise a selectable gene which makes it possible to select or identifythe infected cells. There may be mentioned the genes neo (encodingneomycin phosphotransferase) conferring resistance to the antibioticG418, dhfr (Dihydrofolate Reductase), CAT (Chloramphenicol Acetyltransferase), pac (Puromycin Acetyl-Transferase) or gpt (XanthineGuanine Phosphoriboxyl Transferase). In general, the selectable genesare known to a person skilled in the art.

Elements necessary for the expression of a gene of interest in a hostcell are understood to mean all the elements allowing its transcriptioninto RNA and the translation of an mRNA into a protein. Among these, thepromoter is of particular importance. In the context of the presentinvention, it may be derived from any gene of eukaryotic or even viralorigin and may be constitutive or regulatable. Moreover, it may bemodified so as to improve the promoter activity, suppress atranscription-inhibiting region, make a constitutive promoterregulatable or vice versa, introduce a restriction site and the like.Alternatively, it may be the natural promoter of the gene to beexpressed. There may be mentioned, by way of examples, the CMV(Cytomegalovirus) viral promoter, the RSV (Rous Sarcoma Virus) viralpromoter, the promoter of the HSV-1 virus TK gene, the early promoter ofthe SV40 virus (Simian Virus 40), the adenoviral MLP promoter or theeukaryotic promoters of the murine or human genes for PGK (PhosphoGlycerate kinase), MT (metallothionein), α1-antitrypsin and albumin(liver-specific), immunoglobulins (lymphocyte-specific). It is alsopossible to use a tumor-specific promoter (α-fetoprotein AFP, Ido etal., 1995, Cancer Res. 55, 3105-3109; MUC-1; PSA for prostate specificantigen, Lee et al., 1996, J. Biol. Chem. 271, 4561-4568; and flt1specific for endothelial cells, Morishita et al., 1995, J. Biol. Chem.270, 27948-27953).

Of course, a gene of interest in use in the present invention may, inaddition, comprise additional elements necessary for the expression(intron sequence, signal sequence, nuclear localization sequence,transcription terminating sequence, site for initiation of translationof the IRES type and the like) or for its maintenance in the host cell.Such elements are known to persons skilled in the art.

The invention also relates to a method of preparing an adenovirusaccording to the invention, according to which

the genome of said adenovirus is transfected into an appropriate cellline,

said transfected cell line is cultured under appropriate conditions inorder to allow the production of said adenovirus, and

said adenovirus is recovered from the culture of said transfected cellline and, optionally, said adenovirus is substantially purified.

The choice of the cell line depends on the deficient functions in theadenovirus according to the invention and a complementation line capableof providing in trans the deficient function(s) will be used. The 293line is suitable for complementing the E1 function (Graham et al., 1977,J. Gen. Virol. 36, 59-72). For a double deficiency E1 and E2 or E4, itis possible to use a line among those described in French patentapplication 96 04413. It is also possible to use a helper virus tocomplement the defective adenovirus according to the invention in anyhost cell or a mixed system using complementation cell and helper virusin which the elements are dependent on each other. The means forpropagating a defective adenovirus are known to a person skilled in theart who may refer, for example, to Graham and Prevec (1991, Methods inMolecular Biology, vol. 7, p. 190-128; Ed. E. J. Murey, The Human PressInc.). The adenoviral genome is preferably reconstituted in vitro inEscherichia coli (E. coli) by ligation or by homologous recombination(see for example French application 94 14470). The methods ofpurification are described in the state of the art. There may bementioned the density gradient centrifugation technique.

The present invention also relates to a cell line comprising, either ina form integrated into the genome or in the form of an episome, a DNAfragment encoding a fiber according to the invention, placed under thecontrol of the elements allowing its expression. Said line may, inaddition, be capable of complementing an adenovirus deficient for one ormore functions selected from those encoded by the E1, E2, E4 and L1-L5regions. It is preferably derived from the 293 line. Such a line may beuseful for the preparation of an adenovirus whose genome lacks all orpart of the sequences encoding the fiber (so as to produce anonfunctional fiber). The subject of the present invention is also thecorresponding method, according to which:

the genome of said adenovirus is transfected into a cell line accordingto the invention,

said transfected cell line is cultured under appropriate conditions inorder to allow the production of said adenovirus, and

said adenovirus is recovered from the culture of said transfected cellline and, optionally, said adenovirus is substantially purified.

The present invention also covers a host cell infected with anadenovirus according to the invention or capable of being obtained by amethod according to the invention. This is advantageously a mammaliancell and, in particular, a human cell. It may be a primary or tumor celland of any origin, for example of hematopoietic origin (totipotent stemcell, leukocyte, lymphocyte, monocyte or macrophage and the like),muscle (satellite cell, myocyte, myoblast, smooth muscle cell), cardiac,nasal, pulmonary, tracheal, hepatic, epithelial or fibroblast origin.

The subject of the invention is also a pharmaceutical compositioncomprising, as therapeutic or prophylactic agent, a host cell or anadenovirus according to the invention or capable of being obtained by amethod according to the invention, in combination with apharmaceutically acceptable carrier. The composition according to theinvention is, in particular, intended for the preventive or curativetreatment of diseases such as genetic diseases (hemophilia, cysticfibrosis, diabetes, Duchenne's. myopathy or Becker's myopathy and thelike), cancers, such as those induced by oncogenes or viruses, viraldiseases, such as hepatitis B or C and AIDS (acquired immunodeficiencysyndrome resulting from HIV infection), recurring viral diseases, suchas viral infections caused by the herpesvirus and cardiovasculardiseases including restenoses.

A pharmaceutical composition according to the invention may bemanufactured conventionally. In particular, a therapeutically effectivequantity of the therapeutic or prophylactic agent is combined with acarrier such as a diluent. A composition according to the invention maybe administered by the local, systemic or aerosol route, in particularby the intragastric, subcutaneous, intracardiac, intra-muscular,intravenous, intraperitoneal, intratumor, intrapulmonary, intranasal orintracheal route. The administration may take place in a single dose orrepeated once or several times after a certain time interval. Theappropriate route of administration and the appropriate dosage varyaccording to various parameters, for example, the individual or patientto be treated or the gene(s) of interest to be transferred. Inparticular, the viral particles according to the invention may beformulated in the form of doses of between 10⁴ and 10¹⁴ pfu(plaque-forming units), advantageously 10⁵ and 10¹³ pfu and, preferably,10⁶ and 10¹² pfu. The formulation may also include a diluent, anadjuvant, a pharmaceutically acceptable excipient as well as astabilizer, preservative and/or solubilizer. A formulation in saline,nonaqueous or isotonic solution is particularly suitable for aninjectable administration. It may be provided in liquid or dry form (forexample a lyophilisate and the like) or any other galenic form commonlyused in the pharmaceutical field.

Finally, the present invention relates to the therapeutic orprophylactic use of an adenovirus or of a host cell according to theinvention or of an adenovirus capable of being obtained by a methodaccording to the invention, for the preparation of a medicament intendedfor the treatment of the human or animal body by gene therapy. Accordingto a first possibility, the medicament may be administered directly invivo (for example by intravenous injection, into an accessible tumor,into the lungs by aerosol and the like). It is also possible to adoptthe ex vivo approach which consists in collecting cells from the patient(bone marrow stem cells, peripheral blood lymphocytes, muscle cells andthe like), transfecting or infecting them in vitro according to priorart techniques and readministering them to the patient.

The invention also extends to a method of treatment according to which atherapeutically effective quantity of an adenovirus or of a host cellaccording to the invention is administered to a patient requiring such atreatment.

EXAMPLES

The following examples illustrate only an embodiment of the presentinvention.

The constructs described below are prepared according to general geneticengineering and molecular cloning techniques detailed in Maniatis etal., (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press,Cold Spring Harbor, N.Y.) or according to the manufacturer'srecommendations when a commercial kit is used. The cloning steps usingbacterial plasmids are preferably carried out in the E. coli 5K (Hubacekand Glover, 1970, J. Mol. Biol. 50, 111-127) or BJ 5183 (Hanahan, 1983,J. Mol. Biol. 166, 557-580) strain. This latter strain is preferablyused for the homologous recombination steps. The strain NM522(Strategene) is suitable for the propagation of the M13 phage vectors.The PCR amplification techniques are known to persons skilled in the art(see for example PCR Protocols-A guide to methods and application, 1990,edited by Innis, Gelfand, Sninsky and White, Academic Press Inc). Asregards the repair of the restriction sites, the technique used consistsin filling the protruding 5′ ends with the aid of the large fragment ofE. coli DNA polymerase I (Klenow). The Ad5 nucleotide sequences arethose used in the Genebank data bank under the reference M73260.

As regards the cell biology, the cells are transfected according tostandard techniques well known to persons skilled in the art. There maybe mentioned the calcium phosphate technique (Maniatis et al., supra),but any other protocol may also be used, such as the DEAE dextrantechnique, electroporation, methods based on osmotic shocks,microinjection or methods based on the use of cationic lipids. As forthe culture conditions, they are conventional. In the examples whichfollow, the human line 293 (ATCC CRL1573) and the murine lines Swiss 3T3(ATCC CCL92), NR6 (Wells et al., 1990, Science 247: 962-964), NR6-hEGFR(Schneider et al., 1986, Proc. Natl. Acad. Sci. USA 83, 333-336), DaudiHLA− (ATCC CCL213) and Daudi HLA+ (Quillet et al., 1988, J. Immunol.141, 17-20) are used. It is indicated that the Daudi line is establishedfrom a Burkitt lymphoma and is naturally deficient in the expression ofβ2-microglobulin and, as a result, does not possess the class I HLAmolecules at its surface (Daudi HLA−). The Daudi-derived cell line E8.1was generated by transfection of a gene encoding β2-microglobulin inorder to restore the expression of class I HLA molecules at theirsurface (Daudi-HLA+; Quillet et al., 1988, J. Immunol. 141, 17-20). Itis understood that other cell lines may also be used.

Example 1 Construction of an Adenovirus Exhibiting a Host Tropism Towardthe Cells Expressing the GRP (for gastrin releasing peptide) Receptor

A. Insertion of the Sequences Encoding the GRP Ligand (GRP fiber).

The plasmid pTG6593 is derived from p poly II (Lathe et al., 1987, Gene57, 193-201) by introducing the complete gene encoding the Ad5 fiber inthe form of an EcoRI-SmaI fragment (nucleotides (nt) 30049 to 33093).The HindIII-SmaI fragment (nt 31994-33093) is isolated and cloned intoM13TG130 (Kieny et al., 1983, Gene 26, 91-99) digested with these sameenzymes, in order to give M13TG6526. The latter is subjected tosite-directed mutagenesis with the aid of the oligonucleotide oTG7000(SEQ ID NO: 2) (Sculptor kit, in vitro mutagenesis, Amersham) in orderto introduce an adaptor encoding a spacer arm of 12 amino acids havingthe sequence PSASASASAPGS (SEQ ID NO: 45). The mutated vector thusobtained, M13TG6527, is subjected to a second mutagenesis which makes itpossible to introduce the sequence encoding the 10 residues of the GRPpeptide (GNHWAVGHLM (SEQ ID NO: 46); Michael et al., 1995, Gene Ther. 2,660-668). The oligonucleotide oTG7001 (SEQ ID NO: 3) is used to thiseffect. The HindIII-SmaI fragment is isolated from the mutated phageM13TG6528 and introduced by the homologous recombination technique(Chartier et al., 1996, J. Virol. 70, 4805-4810) into the plasmidpTG6590 carrying the Ad5 adenoviral genome fragment extending from nt27081 to 35935 and linearized with MunI (nt 32825). The SpeI-ScaIfragment (carrying nt 27082 to 35935 of the Ad5 genome which aremodified by introduction of the spacer arm and of the GRP peptide) isisolated from the preceding vector designated pTG8599 and then exchangedfor the equivalent fragment of pTG6591 previously digested with thesesame enzymes. As a guide, pTG6591 comprises the wild-type adenoviralsequences from positions 21562 to 35935. pTG4600 is obtained from whichthe BstEII fragment (nt 24843 to 35233) is isolated. After homologousrecombination with the plasmid pTG3602 which comprises the Ad5 genome(described in greater detail in international application WO96/17070),the vector pTG4601 is generated.

A cassette allowing the expression of the LacZ gene is introduced inplace of the E1 adenoviral region by homologous recombination betweenthe plasmid pTG4601 linearized with ClaI and a fragment BsrGI-PstIcomprising the LacZ gene encoding β-galactosidase under the control ofthe Ad2 MLP promoter and the SV40 virus polyadenylation signal. Thisfragment is isolated from the vector pTG8526 containing the 5′ end ofthe viral genomic DNA (nt 1 to 6241) in which the E1 region (nt 459 to3328) is replaced with the LacZ expression cassette. Its construction iswithin the capability of persons skilled in the art. The final vector isdesignated pTG4628.

The corresponding viruses AdTG4601 and AdTG4628 are obtained bytransfection of the adenoviral fragments liberated from the plasmidsequences by PacI digestion into the 293 line. As a guide, AdTG4601carries the complete Ad5 genome in which the gene for the fibercomprises at its 3′ end a spacer arm followed by the GRP peptide. Therecombinant virus AdTG4628 carries, in addition, the cassette forexpression of the LacZ reporter gene under the control of the MLPadenoviral promoter.

B. Study of the Tropism of the Virus Carrying the GRP Fiber.

The presence of the GRP peptide in the adenoviral fiber makes itpossible to target the cells expressing at their surface the GRPreceptor. The expression of the messengers encoding the latter isstudied in the 293 cells and in the murine Swiss-3T3 cells (Zachary etal., 1985, Proc. Natl. Acad. Sci. USA. 82, 7616-7620) by Northernblotting. There is used, as probe, a mixture of 2 DNA fragmentscomplementary to the sequence encoding the GRP receptor which arelabeled by conventional techniques with the ³²p isotope. As a guide, thefragments are produced by reverse PCR from total cell RNAs with the aidof the oligonucleotides oTG10776 (SEQ ID NO: 4) and oTG10781 (SEQ ID NO:5) (Battey et al., 1991, Proc. Natl. Acad. Sci. USA 88, 395-399; Corjayet al., 1991, J. Biol. Chem. 266, 18771-18779). The intensity of themRNAs detected is much higher in the case of the Swiss-3T3 cells than inthe 293 cells, indicating the overexpression of the GRP receptor by themurine line.

Competition experiments are carried out on the 2 types of cells. Thecompetitor consists of the head of the Ad5 fiber produced in E. coliwhose adenoviral cellular-receptor binding properties have been shown(Henry et al., 1994, J. Virol 68, 5239-5246). The monolayer cells arepreviously incubated for 30 minutes in the presence of PBS or ofincreasing concentrations of recombinant Ad5 head (0.1 to 100 μg/ml) inDMEM medium (Gibco BRL) supplemented with 2% fetal calf serum (FCS).Next, the virus AdTG4628 whose fiber contains the GRP peptide is addedat a multiplicity of infection of 0.001 infectious unit/cell for 24 h at37° C. The recombinant virus AdLacZ (Stratford-Perricaudet et al., 1992,J. Clin. Invest. 90, 626-630) which carries a native gene for the fiber,is used as control and based on the same experimental conditions. Thecells are then fixed and the expression of the LacZ gene evaluated(Sanes et al., 1986, EMBO J. 5, 3133-3142). The number of blue cells isrepresentative of the efficiency of the viral infection. An inhibitionby competition results in a reduction in the number of colored cellscompared with a control without competitor (PBS).

The addition of recombinant Ad5 head at a concentration of 100 μg/mlstrongly inhibits the infection of the 293 cells by the AdLacZ andAdTG4628 viruses (inhibition level of 95 and 98%). This suggests thatthe presence of the competitor prevents the interaction of theadenoviral fiber with its natural cellular receptor. On the other hand,the two viruses have a different behavior on the Swiss-3T3 cells. Theinfection of the AdTG4628 virus in the presence of 100 μg/ml ofcompetitor is only partially inhibited whereas, under the sameexperimental conditions, that of the AdLacZ virus having the nativefiber is completely inhibited. These results suggest that the infectionof the Swiss-3T3 cells with AdTG4628 is partly mediated by anindependent receptor, probably the GRP receptor which these cellsoverexpress. In conclusion, the addition of the GRP ligand to theC-terminal end of the fiber promotes the infection of the cellsexpressing the GRP receptor independently of the fiber-natural cellularreceptor interaction.

Example 2 Construction of an Adenovirus Exhibiting a Host Tropism Towardthe Cells Expressing the EGF (Epidermal Growth Factor) Receptor

This example describes a fiber carrying the EGF sequences at itsC-terminal end. For that, the oligonucleotides oTG11065 (SEQ ID NO: 6)and oTG11066 (SEQ ID NO: 7) are used to amplify an HindIII-XbaI fragmentfrom the plasmid M13TG6527. The oligonucleotides oTG11067 (SEQ ID NO: 8)and oTG11068 (SEQ ID NO: 9) make it possible to generate an XhoI-SmaIfragment (going from the stop codon up to nt 33093) from M13TG6527. TheDNA complementary to EGF, obtained from ATCC (#59957), is amplified inthe form of an XhoI-XbaI fragment with the aid of the oligonucleotidesoTG11069 (SEQ ID NO: 10) and oTG11070 (SEQ ID NO: 11). The 3 fragmentsdigested with the appropriate enzymes are then religated in order togive an HindIII-SmaI fragment containing the EGF fused to the C-terminalend of the fiber. The same homologous recombination procedure as thatdescribed in Example 1 is applied in order to replace this fragment inits genomic context.

However, it is possible to simplify the cloning steps by introducing aunique BstBI site into the targeted region by conventional mutagenesistechniques. pTG46009 and pTG4213 are obtained with LacZ. The homologousrecombination between pTG4609 linearized with BstBI and the precedingHindIII-SmaI fragment generates the plasmid pTG4225 carrying thewild-type E1 region. Its equivalent carrying the LacZ expressioncassette pTG4226 is obtained by homologous recombination with pTG4213digested with BstBI. The viruses AdTG4225 and AdTG4226 may be producedconventionally by transfection of an appropriate cell line for exampleoverexpressing the EGF receptor.

To test the specificity of infection by these viruses, it is possible touse the NR6 murine fibroblast cells and the NR6-hEGFR cells expressingthe human EGF receptor. Competitions with the recombinant Ad5 head orwith EGF make it possible to evaluate the involvement of the naturalcellular receptors and EGF for mediating infection by the viruses.

Example 3 Modifications of the Head of the Fiber in Order to Eliminatethe Binding to the Natural Cellular Receptor

The mutation of the region of the adenoviral fiber involved in theinteraction with the natural cellular receptor was undertaken in orderto eliminate the capacity of the fiber to bind its natural receptor andthe addition of a ligand will make it possible to modify the tropism ofthe corresponding adenoviruses.

The sequences of the Ad5 fiber encoding the region extending fromresidues 443 to 462 and 529 to 536 were subjected to various mutations.The deletion of the D sheet uses the mutagenesis oligonucleotide oTG7414(SEQ ID NO: 12) and the deletion of the CD loop the oligonucleotide oTGA(SEQ ID NO: 13). The oligonucleotide oTGB (SEQ ID NO: 14) allows, forits part, the deletion of the CD loop and of the D sheet. Theoligonucleotide OTG 7416 (SEQ ID NO: 38) allows the deletion of the Hsheet. All these oligonucleotides contain a BamHI site which makes itpossible to easily detect the mutants and, also, to insert the sequencesencoding a ligand, for example the EGF peptide.

Another series of modifications consists in replacing these deletedregions with the equivalent sequences derived from the Ad3 fiber (D+CD5to D+CD3 means that the CD and D region of the Ad5 fiber is replaced byits equivalent from Ad3). Indeed, many data show that Ad5 and Ad3 do notbind to the same receptor, so that such a substitution should abolishthe infection mediated by the Ad5 receptor and target the cells carryingthe Ad3 receptor. The replacement of the Ad5 CD loop with that of Ad3uses oTG11135 (SEQ ID NO: 15), the replacement of the D sheet of the Ad5fiber with that of the Ad3 fiber is carried out by the oligonucleotideoTG10350 (SEQ ID NO: 16) and the replacement of the D sheet and of theCD loop of Ad5 with those of Ad3 is carried out on the preceding mutantwith the aid of oTG11136 (SEQ ID NO: 17). The replacement of the H sheetis carried out with the aid of the oligonucleotide oTG10352 (SEQ ID NO:39).

This target region of the adenoviral head was also modified by a seriesof point mutations:

replacement from the αα GSLA elbow to the αα DKLT elbow: oTGC (SEQ IDNO: 18),

replacement from the αα SGTV elbow to the αα DKLT elbow: oTGD (SEQ IDNO: 19),

G443 to D (G443D): oTGE (SEQ ID NO: 20),

L445 to F (L445F) oTGF (SEQ ID NO: 21),

G450 to N (G450N): oTGG (SEQ ID NO: 22),

T451 to K (T451K): oTGH (SEQ ID NO: 23),

V452 to N (V425N): oTGI (SEQ ID NO: 24),

A 455 to F (A455F): oTGJ (SEQ ID NO: 25),

L457 to K (L457K): oTGK (SEQ ID NO: 26),

I459 to A (I459A): oTGL (SEQ ID NO: 27).

The oTGEs to I introduce mutations in the CD loop of the adenoviralfiber on amino acids which are nonconservative between Ad5 and Ad3whereas the oTGJs to K relate to amino acids of the D sheet which arenot engaged in a hydrogen bond stabilizing the structure.

The mutageneses may be carried out on the vector M13TG6526 or M13TG6528.The first carries the wild-type HindIII-SmaI fragment and the secondthis same fragment modified by insertion of the GRP sequences. Theplasmids carrying the adenoviral genome may be reconstituted asdescribed above for the plasmids pTG4609 (wild-type E1) and pTG4213(LacZ in place of the E1 region). The viruses are generated bytransfection of the 293 cells or of cells overexpressing the receptorbinding the relevant ligand. Such cells may be generated by transfectionof the corresponding complementary DNA. Cells are preferably used whichdo not naturally express the natural cellular receptor for adenoviruses,for example the Daudi line (ATCC CCL213).

The viability of the various mutants is evaluated by transfection ofcells 293 and 293-Fb+ (293 cells transfected with a vector forexpressing the wild-type Ad5 fiber). However, the transfectionefficiency is variable from one experiment to another, even as regardsan adenovirus carrying a wild-type fiber having incorporated the GRPpeptide at its C-terminal end (AdFbGRP). To standardize the results, theplaques obtained after transfection of the 293-Fb+ cells are first ofall amplified on this same line and the viruses generated are titrated:at this stage, they carry either the wild-type fiber or the mutant fiberor both types. The 293 cells are then infected with these viruses at alow multiplicity of infection which makes it possible to infect onlyabout 10% of the cells (MOI of about 0.2 infectious unit/cell) and theextent of the viral infection is determined at 7 days post-infection bymeasuring the accumulation of the viral DNA and the viral titer. Thepropagation of the infection being dependent on the mutated fiber, theadvantageous mutants are those which do not give rise to a productiveinfection, showing that the mutation alters the binding to the naturalreceptor. The propagation capacity of the adenoviruses carrying themutated fiber is compared with that of a wild-type adenovirus and of anAdFbGRP.

The results show that the insertion of the GRP peptide at the end of thewild-type fiber slightly reduces the growth of the corresponding viruscompared with a wild-type Ad, but the multiplication factor isnevertheless of the order of 1000. The mutations V452N, D+CD5 to D+CD3and L445F significantly reduce (factor of 3 to 11) the affinity of themutated fiber head for the natural cellular receptor for adenoviruses.The mutations ΔD (deletion of the D sheet of the Ad5 fiber), ΔCD+D(deletion of the CD loop and of the D sheet), CD5 to CD3 (replacement ofthe CD loop of an Ad5 with its equivalent from an Ad3), G443D, A455F,L457K and I459A abolish the propagation of the corresponding viruses inthe 293 cells (multiplication factor less than 1).

Next, the capacity of the mutant viruses to penetrate into the targetcells by means of the GRP receptor is verified. The advantageous mutantsare those which exhibit a significant multiplication factor (greaterthan 1). In this regard, it is possible to use a line designated293-GRPR expressing MHC-I and the GRP receptor at high levels. It isgenerated by transfecting the 293 cells with a eukaryotic expressionplasmid carrying the cDNA for the GRP receptor (Corjay et al., 1991, J.Biol. Chem. 266, 18771-18779). The expression cassette consists of theearly CMV promoter (Boshart et al., 1985, Cell 41, 521), the sequencesfor splicing the β-globin gene, the cDNA encoding the GRP receptor andthe polyA sequences of the β-globin gene. The selection of thetransformants is carried out in the presence of hygromycin (350 μg/ml)and 50 clones are selected, amplified and tested for the expression ofthe mRNA encoding the receptor by the Northern technique. The mostproductive clones are assembled and designated 293-GRPR. The expressionof the protein may also be checked by FACS with the aid of a GRP peptideconjugated with biotin and avidin-FITC followed by detection with theaid of fluoroscein.

Example 4 Insertion of the Ligand into a Capsid Protein Other Than theFiber in Combination with one of the Abovementioned Modifications of theFiber

This example describes the insertion of the EGF ligand into the hexoncapsid protein. Of course, it is preferable for the correspondingadenovirus to have lost its capacity for attachment to the naturalcellular receptor. Its genome may for example include a modified fibergene (see Example 3) or may lack at least part of the sequences of thefiber.

A transfer plasmid is constructed for the homologous recombinationcovering the region of the Ad5 genome encoding the hexon (nt18842-21700). The Ad5 HindIII-XhoI fragment (nt 18836-24816) is clonedinto pBSK+ (Strategene) digested with these same enzymes in order togive the plasmid pTG4224. The sequences encoding the EGF peptide areintroduced into the hypervariable L1 loop of the hexon by creatingchimeric fragments by PCR: hexon (nt19043-19647)-XbaI-EGF-BsrGI-hexon(nt19699-20312). The nt19043 to 19647 fragment is obtained by PCRamplification from the plasmid pTG3602 with the oligonucleotidesoTG11102 (SEQ ID NO: 28) and oTG11103 (SEQ ID NO: 29). The nt19699 to20312 fragment is amplified from the same DNA with the oligonucleotidesoTG11104 (SEQ ID NO: 30) and oTG11105 (SEQ ID NO: 31). The EGF is clonedfrom the cDNA with the aid of the oligonucleotides oTG11106 (SEQ ID NO:32) and oTG11107 (SEQ ID NO: 33) making it possible to place the codingsequence of the EGF in phase with the hexon. The PCR products aredigested with the appropriate enzymes and then religated. The chimericfragment may then be inserted by homologous recombination into theplasmid pTG4224 linearized with NdeI (nt 19549), to give pTG4229. Thesequences encoding the modified hexon may be obtained by HindIII-XhoIdigestion and replaced in their genomic context by homologousrecombination. The vector pTG3602, pTG4607, pTG4629 linearized with SgfIor a vector carrying the adenoviral genome deleted for the fibersequences (such as pTG4607 described below) or expressing a modifiedfiber in accordance with Example 3 may be used.

The adenoviral genome incapable of producing a functional native fiberis obtained by a deletion affecting the initiator codon but notextending to the other adenoviral ORFs. The procedure is carried out inthe following manner: the adenoviral fragment in 5′ of the deletion (nt30564 to 31041) is amplified by PCR with the aid of the primers oTG7171and oTG7275 (SEQ ID NO: 34 and 35). The amplification of the fragment in3′ (nt 31129 to 33099) uses the primers oTG7276 and oTG7049 (SEQ ID NO:36 and 37). The PCR fragments are digested with XhoI and ligated beforebeing introduced by homologous recombination into the vector pTG6591linearized with NdeI, to give pTG4602. Next, the BstEII fragmentisolated from the latter is subjected to a homologous recombination withthe vector pTG3602 digested with SpeI. pTG4607 is obtained. The vectorpTG4629 is equivalent to pTG4607, but carries, in addition, the LacZexpression cassette in place of E1.

The corresponding viruses may be obtained after transfection of 293 or293-Fb+ cells or of cells overexpressing the EGF receptor. The study ofthe specificity of infection may be carried out as previously describedusing EGF as competitor.

Example 5 Construction of a Mutant of the Fiber Deleted for the E and FSheets

A deletion mutant for the EF sheets of the domain of the head of the Ad5fiber was also generated. The plasmid pTG6593 is digested with HindIIIand SmaI and the fragment carrying the fiber sequences is isolated andcloned into the vector M13TG130 cleaved with HindIII and SmaI. Thedeleterious mutation uses the oligonucleotide indicated in SEQ ID NO:40. The mutated HindIII-SmaI fragment is recombined in E. coli withpTG4609 linearized with BstBI (Chartier et al., 1996, J. Virol. 70,4805-4810). The latter contains the complete Ad5 genome containing aBstBI site at position 32940 downstream of the stop codon of the fiber.

The trimerizing capacity of the modified fiber is tested on NDS-PAGE gel(Novelli and Boulanger, J. of Biological Chemistry, 1991, 266,9299-9303) using the protein produced by the recombinant route in theinsect cells Sf9 with the aid of a recombinant baculovirus carrying thecorresponding sequences placed under the control of the polyhedrinpromoter. In parallel, the viability (or propagating capacity) of thevirions carrying the mutated fiber is determined by transfection of the293 and 293Fb+ cells. Finally, the binding of the fiber to the MHC-I andCAR cellular receptors may be studied in competition experiments forinfection by a recombinant adenovirus Ad5-Luc on Daudi-HLA+ and CHO-CARcells (Bergelson et al., 1997, Science 275, 1320-1323). As a guide, thevirus Ad5Luc is a replication-competent adenovirus which contains theluciferase gene placed under the control of the SV40 virus (Simian Virus40) early promoter inserted into the E3 region of the adenoviral genome(Mittal et al., 1993, Virus Research 28, 67-90).

The ΔEF fiber accumulates in the form of trimers, can be propagated in293 and 393Fb+ cells and is not a competitor for the infection ofDaudi-HLA+ cells with Ad5Luc. It is capable of partially inhibiting theinfection of the CHO-CAR cells, but less efficiently than the wild-typefiber. This assumes that the E and F sheets are important for theattachment of Ad5 to the MHC-1 receptor whereas it plays a more minorrole, perhaps of stabilization in the binding to CAR. The insertion of anew ligand should make it possible to redirect the infectivity towardthe cells carrying the receptor recognized by the ligand.

46 1 581 PRT Ad5 fiber Mastadenovirus 1 Met Lys Arg Ala Arg Pro Ser GluAsp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly ProPro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly PheGln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro LeuVal Thr Ser Asn Gly Met Leu Ala Leu 50 55 60 Lys Met Gly Asn Gly Leu SerLeu Asp Glu Ala Gly Asn Leu Thr Ser 65 70 75 80 Gln Asn Val Thr Thr ValSer Pro Pro Leu Lys Lys Thr Lys Ser Asn 85 90 95 Ile Asn Leu Glu Ile SerAla Pro Leu Thr Val Thr Ser Glu Ala Leu 100 105 110 Thr Val Ala Ala AlaAla Pro Leu Met Val Ala Gly Asn Thr Leu Thr 115 120 125 Met Gln Ser GlnAla Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile 130 135 140 Ala Thr GlnGly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln 145 150 155 160 ThrSer Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr 165 170 175Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu 180 185190 Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195200 205 Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr210 215 220 Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys ValThr 225 230 235 240 Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln LeuAsn Val Ala 245 250 255 Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg LeuIle Leu Asp Val 260 265 270 Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu AsnLeu Arg Leu Gly Gln 275 280 285 Gly Pro Leu Phe Ile Asn Ser Ala His AsnLeu Asp Ile Asn Tyr Asn 290 295 300 Lys Gly Leu Tyr Leu Phe Thr Ala SerAsn Asn Ser Lys Lys Leu Glu 305 310 315 320 Val Asn Leu Ser Thr Ala LysGly Leu Met Phe Asp Ala Thr Ala Ile 325 330 335 Ala Ile Asn Ala Gly AspGly Leu Glu Phe Gly Ser Pro Asn Ala Pro 340 345 350 Asn Thr Asn Pro LeuLys Thr Lys Ile Gly His Gly Leu Glu Phe Asp 355 360 365 Ser Asn Lys AlaMet Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp 370 375 380 Ser Thr GlyAla Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr 385 390 395 400 LeuTrp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu 405 410 415Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile 420 425430 Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile 435440 445 Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn450 455 460 Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp AsnPhe 465 470 475 480 Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr AsnAla Val Gly 485 490 495 Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser HisGly Lys Thr Ala 500 505 510 Lys Ser Asn Ile Val Ser Gln Val Tyr Leu AsnGly Asp Lys Thr Lys 515 520 525 Pro Val Thr Leu Thr Ile Thr Leu Asn GlyThr Gln Glu Thr Gly Asp 530 535 540 Thr Thr Pro Ser Ala Tyr Ser Met SerPhe Ser Trp Asp Trp Ser Gly 545 550 555 560 His Asn Tyr Ile Asn Glu IlePhe Ala Thr Ser Ser Tyr Thr Phe Ser 565 570 575 Tyr Ile Ala Gln Glu 5802 60 DNA Artificial sequence Synthetic oligonucleotide 0TG7000 (codesfor PSASASASAPGS) 2 aacgattctt tagctgccgg gagcagaggc ggaggcggaggcgctgggtt cttgggcaat 60 3 57 DNA Artificial sequence Syntheticoligonucleotide otg7001 (codes for GRP). 3 aacgattctt tacatcaggtggcccacagc ccagtggttt ccgctgccgg gagcaga 57 4 20 DNA Artificial sequenceSynthetic oligonucleotide oTG10776 4 ccttccacgg gaagattgta 20 5 20 DNAArtificial sequence Synthetic oligonucleotide oTG10781 5 ggggtgtctgtcttcacact 20 6 26 DNA Artificial sequence Synthetic oligonucleotideoTG11065 6 gggaagcttg aggttaacct aagcac 26 7 28 DNA Artificial sequenceSynthetic oligonucleotide oTG11066. 7 gggtctagag ctgccgggag cagaggcg 288 29 DNA Artificial sequence Synthetic oligonucleotide oTG11067. 8gggctcgagt tatgtttcaa cgtgtttat 29 9 24 DNA Artificial sequenceSynthetic oligonucleotide oTG11068. 9 gtgcccgggg agtttattaa tatc 24 1031 DNA Homo sapiens 0TG11069 10 gcgtctagaa atagtgactc tgaatgtccc c 31 1146 DNA Homo sapiens oTG11070 11 gcgctcgagc acaaacgatt ctttagcgcagttcccacca cttcag 46 12 42 DNA Mastadenovirus 0TG7414 12 tagcactccattttcgtcgg atccttgaac tgttccagat at 42 13 43 DNA Mastadenovirus oTGA 13cttataataa gatgagcact ggatccagcc aaaactgaaa ctg 43 14 45 DNAMastadenovirus oTGB 14 gtagcactcc attttcgtcg gatccaacag ccaaaactga aactg45 15 88 DNA Mastadenovirus oTG11135 15 cgtcaaatct tataataaga tgagcactcacgtttttgtt tttaaacagg gtgttgtagt 60 cgctaacagc caaaactgaa actgtagc 88 1664 DNA Mastadenovirus oTG10350 16 gtagcactcc attttcgtca aagtagagctccacgttgat actttgaact gttccagata 60 ttgg 64 17 88 DNA MastadenovirusoTG11136 17 cgtcaaagta gagctccacg ttgatactca cgtttttgtt tttaaacagggtgttgtagt 60 cgctaacagc caaaactgaa actgtagc 88 18 56 DNA MastadenovirusoTGC 18 ttgaactgtt ccagatattg gggtcagttt gtctttaaca gccaaaactg aaactg 5619 48 DNA Mastadenovirus oTGD 19 aataagatga gcactttggg tcagtttgtctattggagcc aaactgcc 48 20 44 DNA Mastadenovirus oTGE 20 ccagatattggagccaaact gtctttaaca gccaaaactg aaac 44 21 42 DNA Mastadenovirus oTGF21 tgttccagat attggagcga aactgccttt aacagccaaa ac 42 22 42 DNAMastadenovirus oTGG 22 atgagcactt tgaactgtgt tagatattgg agccaaactg cc 4223 44 DNA Mastadenovirus oTGH 23 taagatgagc actttgaacc tttccagatattggagccaa actg 44 24 45 DNA Mastadenovirus oTGI 24 cttataataagatgagcact ttggtttgtt ccagatattg gagcc 45 25 48 DNA Mastadenovirus oTGJ25 gtcaaatctt ataataagat ggaaactttg aactgttcca gatattgg 48 26 47 DNAMastadenovirus oTGK 26 ccattttcgt caaatcttat aattttatga gcactttgaactgttcc 47 27 46 DNA Mastadenovirus oTGL 27 gcactccatt ttcgtcaaatctagcaataa gatgagcact ttgaac 46 28 23 DNA Mastadenovirus oTG11102 28cggttcatcc ctgtggaccg tga 23 29 38 DNA Mastadenovirus oTG11103 29ggcctctaga gttgagaaaa attgcatttc cacttgac 38 30 23 DNA MastadenovirusoTG11104 30 ggtattgtac agtgaagatg tag 23 31 23 DNA MastadenovirusoTG11105 31 cgttggaagg actgtacttt agc 23 32 38 DNA Homo sapiens oTG1110632 cgcgtctaga ggcgaatagt gactctgaat gtcccctg 38 33 45 DNA Homo sapiensoTG11107 33 ccactgtaca ataccacttt agggcgcagt tcccaccact tcagg 45 34 21DNA Mastadenovirus oTG7171 34 atggttaact tgcaccagtg c 21 35 27 DNAMastadenovirus oTG7275 35 gggctcgagc tgcaacaaca tgaagat 27 36 27 DNAMastadenovirus oTG7276 36 ccgctcgaga ctcctccctt tgtatcc 27 37 20 DNAMastadenovirus oTG7049 37 ctgcccggga gtttattaat 20 38 42 DNAMastadenovirus oTG7416 38 tgtttcctgt gtaccgttgg atcctttagt tttgtctccg tt42 39 64 DNA Mastadenovirus oTG10352 39 tgtttcctgt gtaccgttta gcatcacggtcacctcgaga ggtttagttt tgtctccgtt 60 taag 64 40 42 DNA Mastadenovirus 40tgtataggct gtgccttcgg atccccaata ttctgggtcc ag 42 41 19 PRT Ad5 fiberMastadenovirus 41 Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His LeuIle Ile Thr 1 5 10 15 Arg Phe Asp 42 19 PRT Ad3 fiber Mastadenovirus 42Val Asn Thr Leu Phe Lys Asn Lys Asn Val Ser Ile Asn Val Glu Leu 1 5 1015 Tyr Phe Asp 43 8 PRT Ad5 fiber Mastadenovirus 43 Pro Val Thr Leu ThrIle Thr Leu 1 5 44 8 PRT Ad3 fiber Mastadenovirus 44 Pro Leu Glu Val ThrVal Met Leu 1 5 45 12 PRT Artificial Sequence adaptor encoding a spacerarm of 12 amino acids 45 Pro Ser Ala Ser Ala Ser Ala Ser Ala Pro Gly Ser1 5 10 46 10 PRT Artificial Sequence GRP peptide 46 Gly Asn His Trp AlaVal Gly His Leu Met 1 5 10

What is claimed is:
 1. A method for producing an adenovirus whose genomelacks all or part of the sequences encoding a fiber, comprising:transfecting the genome of said adenovirus into a cell line comprising,either in a form integrated into the genome or in the form of anepisome, a DNA fragment or expression vector encoding an adenovirusfiber modified by substitution mutation of one or more residues of anative fiber, wherein said native fiber residues are directed toward thenatural cellular receptor recognized by the native adenovirus fiber, andwherein said residues are between the CD loop and the β sheet I of saidfiber, the modification not including deletion of the entire knobregion, said DNA fragment or expression vector placed under the controlof the elements allowing its expression in said cell line, culturingsaid transfected cell line under appropriate conditions in order toallow the production of said adenovirus, and recovering said adenovirusfrom the culture of said transfected cell line.
 2. The method of claim1, further comprising substantially purifying said recovered adenovirus.3. An adenovirus made by the process comprising: a) identifying one ormore residues of a natural adenoviral fiber which are directed towardthe natural cellular receptor of a natural adenovirus and which arebetween the CD loop and the β sheet I of the adenoviral fiber, b)modifying said fiber by mutating one or more of said residues so as toeliminate binding to the natural cellular receptor, but not by deletingthe entire knob region, c) either incorporating sequence encoding themodified fiber of step b) into a genome of an adenovirus which lacks afunctional native fiber, or providing the modified fiber in trans in acell line, d) transfecting the genome of said adenovirus into anappropriate cell line, e) culturing said transfected cell line underappropriate conditions in order to allow the production of saidadenovirus, and f) recovering said adenovirus from said culture of saidtransfected cell line.
 4. An adenovirus comprising an adenovirus fiber,modified by substitution mutation of one or more residues of a nativefiber, wherein said native fiber residues are directed toward thenatural cellular receptor recognized by the native adenovirus fiber, andwherein said residues are between the CD loop and the β sheet I of saidfiber and further comprising a ligand inserted at the C-terminal end ofthe fiber, which is capable of recognizing a cell surface moleculedifferent from the natural cellular receptor of said adenovirus in itsnatural form.
 5. The adenovirus of claim 4, wherein said adenovirus is areplication-defective recombinant adenovirus.
 6. The adenovirus of claim5, wherein said adenovirus is deleted for all or part of the E1 region.7. The adenovirus of claim 6, wherein said adenovirus is further deletedfor all or part of the E2, E4 and/or L1-L5 region.
 8. The adenovirus ofclaim 5, wherein said adenovirus comprises a gene of interest selectedfrom the genes encoding a cytokine, a cellular or nuclear receptor, aligand, a coagulation factor, the CFTR protein, insulin, dystrophin, agrowth hormone, an enzyme, and enzyme inhibitor, a polypeptide withantitumor effect, a polypeptide capable of inhibiting a bacterialinfection, a polypeptide capable of inhibiting a parasitic infection, apolypeptide capable of inhibiting a viral infection, a polypeptidecapable of inhibiting HIV, an antibody, a toxin, an immunotoxin and amarker.
 9. The adenovirus of claim 5, wherein said adenovirus is deletedfor all or part of the E3 region.
 10. The adenovirus of claim 4, whereinthe ligand is a polypeptide.